Bone marrow failure and the new telomere diseases: practice and research

Abstract

The telomeropathies are a newly described group of human diseases based on the genetics and molecular biology of the telomeres, the ends of chromosomes. Telomeres are repeated hexanucleotides and their associated proteins; the protect chromosomes from recognition as damaged DNA, and their inevitable gradual loss with DNA replication is harmless as they are noncoding. However, when telomeres become critically short in a cell, senescence, apoptosis, or, rarely malignant transformation results. In individuals with mutations in genes involved in telomere repair, especially the enzymatic telomerase complex, telomere attrition is accelerated. Severe deficiencies result in dyskeratosis congenita, a congenital aplastic anemia with associated mucocutaneous abnormalities. Mutations in TERT, the catalytic component, and TERC, the RNA template, can behave as risk factors for the development of bone marrow failure, pulmonary fibrosis, and hepatic cirrhosis. Both penetrance and organ specificity are variable and not well understood. Chromosome instability is a result of critical shortening of telomeres and cancer. For example, short telomeres are the major prognostic risk factor for clonal evolution to myelodysplasia and acute leukemia. Practically, hematologists need to recognize the multisystem presentation of telomere disease, implications for outcomes, and options for therapy.

Keywords:

• Cirrhosis
• Pulmonary fibrosis
• Aplastic anemia
• Genetics

Telomeres are the hexameric nucleotide sequences that are repeated hundreds to thousands of times (TTAGGG in humans) at the extremity of linear chromosomes, bound to a group of protective proteins, collectively termed shelterin.1 This structure has two critical functions. First, it serves to protect the chromosome from recognition by DNA exonucleases and other harmful enzymes. Second, due to the semiconservative nature of DNA replication, the noncoding character of the telomere sequences ameliorates the consequences of inevitable loss of genetic information at every cell division. Telomere attrition occurs in the cell at mitosis, and telomeres shorten in replicating tissues over the lifespan of an organism. At birth, white blood cells’ telomeres are sufficient in length for about 200 cell divisions. With normal aging of an animal and in cell culture, cells divide and telomeres shorten. When telomeres become critically short, nuclear signaling causes the cell to cease proliferation, and to enter cell senescence or undergo apoptosis. Telomere attrition explains the ‘Hayflick limit’, the number of mitoses a cell is capable of undergoing in vitro. Telomere length is therefore a type of ‘mitotic clock’, a measure of a cell’s proliferative history. Under circumstances in which cell proliferation continues despite critically short telomeres, the telomere’s protective function is lost. Recombination between chromosomes can lead to chromosome instability, aneuploidy, and transformation to a cancer phenotype. Some proliferative cells can elongate telomeres enzymatically. The enzyme telomerase (encoded by the gene TERT) is a reverse transcriptase that employs a small RNA molecule template (encoded by TERC) to extend telomeres. Thus, telomerase counterbalances the effects of cell division and cellular genetic ‘aging’, preventing senescence, apoptosis, and genetic instability.

Telomere Diseases

Dyskeratosis congenita

Telomere repair may be constitutionally defective due to mutations that severely reduce telomerase’s capacity to elongate telomeres, leading to accelerated telomere attrition. Dyskeratosis congenita, a rare inherited bone marrow failure disease, is the classic ‘telomere disease’.2 The inherited defect in X-linked dyskeratosis congenita is due to mutations in a gene named DKC1. DKC1 encodes dyskerin, a protein that binds to the RNA component of telomerase and stabilizes the telomerase complex. Heterozygous mutations in TERC occur in some families with autosomal dominant dyskeratosis congenita. The severe phenotype of X-linked dyskeratosis congenita is due to hemizygous loss of DKC1, and thus markedly reduced telomerase function. In autosomal dominant dyskeratosis congenita, mutations in TERC and other somatic chromosome genes leads to haploinsufficiency, and the retention of a normal, wild-type allele may modify the severity of the clinical presentation.

Dyskeratosis congenita has pathognomonic features. Children may have components of the typical triad of abnormal nails, a hypo- or hyperpigmented reticular cutaneous eruption, and oral leukoplakia. Aplastic anemia usually occurs in the first decade of life. Lungs and the liver also are affected in dyskeratosis congenita, or these organs fail after stem cell transplant performed in order to correct the hematologic disease.

Telomeropathies in adults

Dyskeratosis congenita with typical features can occasionally present late in life, as in a patient with a DKC1 mutation who does not recover blood counts after chemotherapy. However, mutations in TERC and in the enzyme telomerase, encoded by TERT, act as genetic risk factors rather than as genetic determinants for disease. The first TERT mutations found in humans were in adult patients with apparently acquired aplastic anemia. These patients lacked typical physical anomalies, and they did not have a suggestive family history. Penetrance of the phenotype of TERT and TERC mutations is highly variable among and within families: in severity of laboratory abnormalities such as blood counts, the time of onset of symptoms and signs, and the specific organ(s) involved. Within pedigrees, members with the identical mutation may have no or minimal clinical manifestations (often erythrocyte macrocytosis or modest thrombocytopenia), develop aplastic anemia late in life, or suffer pulmonary fibrosis or hepatic cirrhosis; different organ systems may be affected in different family members at different times, and occasional patients have disease in marrow, lung, and liver. Telomerase mutations have been uncovered in a familial pulmonary fibrosis pedigrees,3 and in general populations of patients with severe cirrhosis.4,5

Telomere Shortening and Cancer

Telomere attrition is associated with and likely an etiology of cancer. Patients with dyskeratosis congenita have a 1000-fold risk of tongue cancer and about 100-fold risk of acute myeloid leukemia.6 In aplastic anemia, patients with the shortest telomeres (absent mutations) are four- to fivefold more likely to have their disease undergo malignant transformation to myelodysplasia and leukemia.7 Telomere free ends of chromosomes and aneuploidy can be detected in the patients’ bone marrow in tissue culture years before clonal evolution is clinically apparent.8 Constitutional telomerase gene mutations and rare polymorphisms are present in some patients with acute myeloid leukemia not associated with aplastic anemia, and they almost always occur with aneuploidy as detected by cytogenetics.9 Short leukocyte telomeres predict for the development of cancer in Barrett’s esophagitis and in ulcerative colitis, also in association with chromosome loss, gain, and rearrangement. In these diseases, the mechanism is less certain, because of the unclear relationship between telomere length determinations of blood leukocytes and the status of telomeres in the affected organ. For example, telomere length of leukocytes may represent a biomarker of exposure to reactive oxygen species and therefore, a surrogate assay for chronic inflammation. Active oxygen can damage telomeres, and cells cultured in room air have excessive telomere shortening in comparison to cells cultured at low oxygen tension.

Single nucleotide polymorphisms in the TERT gene have been repeatedly observed as risk factors in genome wide analyses of many cancers. Short leukocyte telomeres increased the risk of all cancers and of cancer fatalities in a large population followed over a decade.10 Circumstantially, telomere attrition is an accompaniment of aging, itself the major risk for cancer. Iatrogenically, secondary hematologic malignancies occur after chemotherapy and radiation, common therapeutic interventions that would be anticipated to lead to marrow stress and accelerate telomere loss in hematopoietic tissue. In a ‘knockout’ mouse model, crosses with reduced telomerase activity combined with diminished p53 surveillance developed a variety of epidermal cancers, unusual in the rodent but typical of humans.

In most malignancies, telomerase gene up regulation or activation of the alternative pathway of telomere repair (based on recombination rather than enzymatic synthesis) is necessary to establish immortality of the cancer. Telomerase is so frequently increased in cancer cells and cell lines as to be considered an appropriate therapeutic target. Critically short telomeres and a chromosome theory of the etiology of cancer are not in conflict with these findings. Most cells in which telomere shortening reaches criticality likely either die or enter senescence. In those few that survive, perhaps due to inadequate monitoring by p53 and related DNA damage response safeguard mechanisms, telomere repair would be subject to powerful selective pressure. Telomere shortening would also provide the equivalent of a mutator phenotype, increasing spontaneous chromosomal aberrations, from numerical changes to structural abnormalities (translocations, insertions, deletions, and telomere associations as end-to-end fusions), and therefore the pool of aberrant cells upon which selection would act.

Implications in the Clinic

The association of telomerase mutations with disease in three disparate organs systems has important practical consequences for patients and physicians. In the family history, attention should be paid to even mild blood count abnormalities in relatives, as well as more severe hematologic disease, especially acute myeloid leukemia. In other organ systems, usually neglected in a standard history, pulmonary fibrosis and hepatic cirrhosis are important clues to the diagnosis of a telomeropathy. The involvement of multiple medical subspecialties can be confusing; some patients have made their own diagnosis from internet searches. In the patient’s own history, suggestive features are chronicity, relatively moderate degrees of pancytopenia and isolated thrombocytopenia or macrocytic anemia, sparing of leukocytes, and hematologic complications during pregnancy, as well as spontaneous abortions. Telomere length of leukocytes can be measured commercially and is a reliable diagnostic marker when length is severely reduced. The TERT and TERC genes can be sequenced, and telomerase function of an abnormal gene product can be tested in the research laboratory. The finding of a telomerase deficit has consequences for prognosis, treatment, and genetic counseling. Although the diagnosis of telomeropathies can be straight forward, there may be difficulties. Some typical families lack known mutations, and telomere length may be normal even in the presence of etiologic nucleotide substitutions. Rare mutations in shelterin genes can produce severe dyskeratosis but do not alter telomerase repair capacity. Regulatory regions of genes, not routinely screened, may be responsible for telomere disease in some cases.

Research Laboratory Questions

Telomerase expression is tightly regulated in the cell; just a few copies of the complex are present in the cell nucleus and it exerts its function during certain specific periods of the cell cycle. The mechanisms that modulate telomerase gene expression, resultant enzymatic activity, and effect telomere elongation are the focus of intensive research. MYC, a proto-oncogene that regulates the expression of many other genes and of cell pluripotency, activates telomerase expression.

Sex hormones also activate telomerase expression in reproductive and non-reproductive organs, such as the bone marrow.11 The promoter region of the telomerase gene contains regulatory sequences that are modulated by intracellular estrogen; cells exposed to androgens (eventually converted intracellularly to estrogens) or estrogens upregulate telomerase expression. In retrospect, the response of patients with aplastic anemia, especially of children with inherited marrow failure, to androgens may be attributable to this mechanism. However, whether higher blood levels of endogenous sex hormones or exposure to exogenous steroids causes telomere elongation is still unknown.

Conclusions

Telomeres and telomere repair are basic molecular processes in cells possessing linear DNA chromosomes. Accelerated telomere attrition due to genetic defects in telomerase and in the shelterin protein genes is etiologic in several human diseases not previously related in the clinic: aplastic anemia, pulmonary fibrosis, and hepatic cirrhosis. The telomeropathies, especially in their milder and more chronic forms, may not be rare and almost certainly are most often unrecognized by physicians. The importance of telomere repair in tissues under regenerative stress is of special interest, particularly the reactive responses of fibrogenesis and adipogenesis and the role of telomere attrition in linking chronic inflammation to cancer in many organs and diseases. Yet, missing are explanations for genotype–phenotype anomalies — highly variable telomerase gene penetrance, organ specificity, and clinical course. Both the genomic architecture modulating telomerase expression and the effect of the organism’s environment on telomere attrition are poorly understood. Drugs or hormones that might modulate telomerase expression and maintain or elongate telomeres would be appealing in the treatment of the telomeropathies and in conditions in which telomere attrition has known medical consequences.